Journal of Food Engineering 81 (2007) 42–53

www.elsevier.com/locate/jfoodeng

Improvement of dough rheology, bread quality and bread shelf-life

by enzymes combination
a,*
P.A. Caballero , M. Gómez a, C.M. Rosell b

a
Escuela Técnica Superior de Ingenierı́as Agrarias, Área de Tecnologı́a de Alimentos, Universidad de Valladolid, Avda, de Madrid, 44, 34004 Palencia, Spain
b
Instituto de Agroqu´ımica y Tecnologı´a de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot, Valencia, Spain
Received 12 April 2006; received in revised form 9 October 2006; accepted 10 October 2006
Available online 28 November 2006

Abstract

Present work seeks to systematically analyse the individual and synergistic effects of some gluten-crosslinking enzymes (transglutamin-
ase, glucose oxidase and laccase), along with polysaccharide and gluten degrading enzymes (alpha-amylase, xylanase and protease), in
breadmaking systems. Except glucose oxidase (GO) and laccase (LAC), enzymes affected significantly to viscoelastic properties of dough.
Results confirmed the strengthening effect exerted by transglutaminase (TG). However, alpha-amylase (AMYL), xylanase (XYL) and
protease (PROT) promoted a similar decrease in all dynamic moduli analysed, particularly after 180 min of incubation. Additi on of
XYL to TG containing samples showed to be an interesting alternative to prevent excessive dough strengthening. Bread quality param-
eters were significantly affected by individual enzyme addition, except when LAC was used. TG diminished loaf specific volume and pro-
vided a finer crumb structure. Polysaccharide degrading enzymes and PROT led to better shape, greater specific volume and void fraction
of loaves. Significant interactions between TG and all the other enzymes except GO, were proved. According to crumb texture evolution
during storage, bread staling increased with TG addition, whilst AMYL, XYL and PROT exhibited a significant antistaling effect.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Enzymes; Wheat flour; Dough rheology; Bread quality; Bread staling

1. Introduction proteins through the formation of large insoluble poly-

Breadmaking quality of wheat flour is largely deter-
mined by the quantity and quality of its proteins. During
dough mixing, wheat flour is hydrated and the gluten pro-
teins are transformed into a continuous cohesive visco-
elastic gluten protein network. Gluten-crosslinking
enzymes can actively contribute to confer the functional
properties to dough. Transglutaminase (TG; protein–glu-
tamine gamma-glutamyltransferase) (EC 2.3.2.13) has
been reported extensively for its ability to crosslink differ-
ent food proteins (Kuraishi, Yamazaki, & Susa, 2001;
Motoki & Nio, 1983; Motoki & Seguro, 1998; Zhu, Rin-
zema, Tramper, & Bol, 1995). When it is used in bread-
making, TG is able to improve the functionality of flour
*
Corresponding author. Tel.: +34 979 108339; fax: +34 979 108302.
E-mail address: pacaball@iaf.uva.es (P.A. Caballero).
mers (Bonet, Caballero, Gomez, & Rosell, 2005; Cabal-
lero, Bonet, Rosell, & Gomez, 2005; Larre´ et al., 2000).
High molecular weight (HMW) glutenins are the most
affected protein fraction (Bauer, Koehler, Wieser, & Schi-
eberle, 2003a; Gerrard et al., 2001; Larre´ et al., 2000;
Rosell, Wang, Aja, Bean, & Lookhart, 2003), but low
molecular weight (LMW) glutenins (Autio et al., 2005), a-
gliadin (Bauer et al., 2003a) or even water extractable
albumins and globulins (Gerrard et al., 2001) have been
also proposed as substrates for TG.
Oxidative enzymes have a strong impact on the dough
thiol–disulphide system and hence, on the properties of
the dough (Goesaert et al., 2005). Glucose oxidase (GO)
(EC 1.1.3.4) is the currently preferred enzyme alternative
to chemical oxidizing agents for bread improvement (Bonet
et al., 2006; Poulsen & Hostrup, 1998). The hydrogen per-
oxide produced during GO reaction promotes the forma-
tion of disulfide linkages in gluten protein and the

0260-8774/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2006.10.007
 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53
gelation of water soluble pentosans (Gujral & Rosell,
2004a; Hoseney & Faubion, 1981; Primo-Mart´ın, Valera,
& Mart´ınez-Anaya, 2003; Vemulapalli & Hoseney, 1998).
Laccase (LAC; p-diphenol oxygen oxidoreductase) (EC
1.10.3.2) is another oxidative enzyme which recently has
attracted a considerable interest in breadmaking. LAC
catalyses the oxidative gelation of feruloylated arabin-
oxylans by dimerization of their ferulic esters (Figueroa-
Espinoza, Morel, & Rouau, 1998; Labat, Morel, & Rouau,
2001). Through the aforementioned mechanisms, gluten-
modifying enzymes may produce beneficial effects during
breadmaking, affecting positively to rheological behaviour
of dough and the quality of final product. The association
of gluten-modifying enzymes with other enzyme principles
has been also proposed (Bolla´ın & Collar, 2004; Caballero,
Gomez, & Rosell, 2006; Collar & Bollain, 2004; Collar &
Bollain, 2005b). Due to their active contribution to fresh
quality enhancement and/or staling prevention of bakery
products, polysaccharide-degrading enzymes have been
usually used for these aims. Among them, amylases (and
concretely alpha-amylase) and pentosanases are some of
most representative. However, reports on the combined
use of strengthening enzymes are limited. On the other
hand, these enzymes act on different protein fractions
(glutenins, gliadins, albumins or globulins) according to
their particular action mechanism, affecting in different
way to the functional properties of bread dough. Present
work seeks to be a systematic study for analysing the indi-
vidual and synergistic effects of gluten crosslinking enzymes
in breadmaking systems. In order to improve their
response, the effect of the aforementioned enzymes was
evaluated in combination with polysaccharide and gluten-
degrading enzymes (alpha-amylase, xylanase and prote-
ase). Rheological behaviour of dough, fresh pan bread
volume, shape, texture and crumb grain characteristics,
as well as the rate of bread staling were analysed for assess-
ing the effects of enzyme treatments.
2. Materials and methods
2.1. Materials
A commercial blend of wheat flours provided by Hari-
nera Castellana (Medina del Campo, Spain) was used in
this study (Table 1). Six commercial enzymes were used:
a glucose-oxidase [Gluzyme Mono 10000 BG (GO)], con-
taining 10,000 glucose oxidase units/g, a pentosanase
[Pentopan Mono BG (XYL)] containing 2500 fungal
xylanase units/g, a laccase [NZ 27011 (LAC)] containing
10,500 phenol oxidase units/g, an amylase [Fungamyl SG
(AMYL)] containing 2500 fungal amylase units/g, a pro-
tease [Flavourzyme 1000 L (PROT)] containing 1000
aminopeptidase units/g (all of them from Novozymes,
Denmark), and transglutaminase (Microbial TGM
Activa WM, TG) containing 100 transglutaminase
units/g, manufactured by Ajinomoto Co. Inc. (Tokyo,
Japan).
43
Table 1
Quality attributes of wheat flour
Flour
Chemical composition
Protein (% d. wt.) 11.00
Ash (% d. wt.) 0.58
Moisture (% d. wt.) 12.16
Consistogram
Water absorption (%) 52.8
Alveogram
Deformation energy (10—4 J) 146
Curve configuration ratio 0.35
Gluten index
Gluten index (%) 94
Dry gluten (%) 9.00
Wet gluten (%) 26.60
Falling number
Time (s) 405
d. wt.: dry weight.
Instant dry yeast and salt employed in breadmaking
process were obtained from the local market. All chemicals
used for analyses were of analytical grade.
3. Dynamic rheological test
Selected dosages of the enzymes GO, XYL, LAC,
AMYL, PROT and TG were added following the sup-
plier’s recommendations, 3 mg, 6 mg, 20 ll, 1 mg, 5 ll
and 500 mg/100 g of flour respectively. Enzymes were
added according to the experimental design showed in
Table 2. All of them were tested at two levels: 0 (absence
of enzyme) and 1 (presence of enzyme at recommended
dose). Flour and enzymes (when added) were mixed during
one hour before the tests, using a Rotary Mixer MR 2L
(Chopin, Tripette et Renaud, France).
Dough was prepared by mixing flour-enzyme blends
with the water [52.8% (w/v), flour basis] in the Alveograph
mixer, according to procedure summarized in the AACC
standard method 54-30 (AACC, 2000). After mixing,
dough was extruded and cut with a knife-spatula in three
pieces that were placed between two glass plates. The pieces
were sheeted to a thickness of 5 mm and cut using a circu-
lar 54 mm diameter cutter. The resulting pieces were placed
in the resting compartment of the Alveograph at 25 °C, and
kept for different resting periods (30, 60 and 180 min),
before testing in the dynamic rheometer.
Dynamic rheological analysis was performed using a
controlled stress rheometer (RheoStress 1, Thermo Haake,
Karlsruhe, Germany) with parallel plate geometry (60 mm
diameter). The dough was placed between parallel plates,
the gap adjusted to 3 mm and the excess dough removed.
To prevent drying at the edges, a thin layer of vaseline
oil was applied to cover the exposed dough surfaces. Before
measurements, doughs rested for 5 min, to allow relaxation
after sample handling. To determine the linear viscoelastic
44 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53

Table 2 0.2 g sodium propionate and the amount of enzyme indi-

Half-fractional factorial design 2 6 for sampling cated previously for each sample. Dough was optimally
Sample no. Factorsa mixed (14 min), divided into 315 g pieces, hand-rounded,
A B C D E F mechanically moulded, put into well-greased tin pans
1 0 0 0 0 0 0
(measuring 195 · 86 mm), and proofed for 90 min at
2 0 1 1 0 1 1 30 °C and 75% RH. The pieces were baked into an elec-
3 0 1 0 0 1 0 tric oven for 35 min at 200 °C. Loaves were removed from
4 0 1 0 1 1 1 the pans, cooled for 2 h at room temperature, then
5 0 1 1 1 1 0
6 0 0 0 1 1 0
packed in plastic bags and stored at 25 °C for aging
7 0 0 1 1 1 1 studies.
8 1 1 1 1 0 0
9 1 0 0 1 1 1 3.2. Evaluation of bread quality
10 1 1 0 1 0 1
11 0 1 1 0 0 0
Quality analysis of fresh bread samples was carried out
12 0 1 0 1 0 0
13 1 1 1 1 1 1 by measuring weight, volume (determined by seed displace-
14 1 0 0 0 0 1 ment in a loaf volume meter), specific volume, and height/
15 0 1 0 0 0 1 width ratio of the central slice.
16 0 0 1 1 0 0 Crumb texture was determined by a Texture Analyzer
17 1 0 1 1 1 0
TA-XT2i (Stable Microsystems, Surrey, UK) provided
18 0 0 0 1 0 1
19 1 0 0 1 0 0 with the software ‘‘Texture Expert’’, and equipped with
20 1 0 1 0 0 0 an aluminium 25 mm diameter cylindrical probe. Slices of
21 1 0 1 0 1 1 2 cm thickness were compressed to 50% of their original
22 1 1 0 1 1 0 height in a ‘‘Texture Profile Analysis’’ double compression
23 1 1 0 0 0 0
test (TPA), at 1 mm/s speed test, with a 30 s delay between
24 1 1 1 0 1 0
25 1 1 0 0 1 1 first and second compression. Primary parameters [hard-
26 1 1 1 0 0 1 ness (gram-force, gf), cohesiveness, springiness and
27 0 0 0 0 1 1 resilience] and secondary mechanical characteristics [gum-
28 0 0 1 0 1 0 miness (gf) and chewiness (gf)] were calculated from the
29 1 0 0 0 1 0
TPA graphic. Bread texture was measured over 12-day per-
30 0 0 1 0 0 1
31 0 1 1 1 0 1 iod of storage.
32 1 0 1 1 0 1 Crumb grain characteristics of bread were assessed
a
Levels (0, 1) of factors (A–F): A = Transglutaminase (TG): none (0), using a digital image analysis (DIA) system. Images were
500 mg/100 g flour (1); B = Glucose oxidase (GO): none (0), 3 mg/100 g previously acquired at 300 dots per inch (0.0843 mm/
flour (1); C = Laccase (LAC): none (0), 20 ll/100 g flour (1); D = Amilase pixel) with a 1236USB Artec scanner (Ultima Electronics
(AMYL): none (0), 1 mg/100 g flour (1); E = Pentosanase (XYL): none Corp., Taiwan). The analysis was performed on
(0), 6 mg/100 g flour (1); F = Protease (PROT): none (0), 20 ll/100 g flour 41 · 41 mm squares taken from the centre of the slice.
(1). This field of view represented approximately one-third
of the cross-sectional area of the loaves. Images were pro-
region of the dough, dynamic moduli were collected and cessed using Leica QWin Pro V3.1 software (Leica Micro-
plotted as a function of the applied stress. systems Imaging Solutions Ltd., UK). A cluster analysis
Oscillatory tests with a frequency sweep from 0.1 to method commonly known as the ‘‘K-means algorithm’’
100 Hz were conducted at a constant stress of 5 Pa at was used to obtain, for each bread slice examined, an
25 °C. The dynamic rheological properties of samples were optimum gray level threshold to divide images into
regions of cells and surrounded cell wall material (Sapir-
assessed by the storage modulus G 0 (elastic modulus) and
stein, 1999). Subsequent to cell detection, feature extrac-
the loss modulus G00 (viscous modulus). The complex mod-
tion was performed for each bread slice analysed. The
ulus (G*) that represents the resistance of dough to defor-
mation or the total energy needed to induce changes in the crumb grain characteristics studied were: crumb bright-
samples was calculated as G* = (G 02 + G002)1/2. To ness (mean gray level), mean cell area (mm 2), cell density
detect significant differences among enzyme treatments, (cells/cm2; higher levels denote finer structure), cell to
the values of dynamic moduli obtained at a frequency of total area ratio (or void fraction, computed as the per-
1 Hz were used (Caballero et al., 2005; Mart´ınez-Anaya centage of the total analysed square occupied by detected
& Jimenez, 1997). cells), mean cell wall thickness (mm; calculated as the
averaged mean intercellular distance of neighbouring cells
3.1. Breadmaking procedure sampled) and crumb grain uniformity (computed as the
ratio of number of small to large cells using a cell area
Dough formulation, based on 100 g flour, included: threshold of 4.0 mm2; larger values denote a more uni-
form cellular structure) (Sapirstein, 1999).
57 ml water, 2 g salt, 0.83 g instant active dry yeast,
 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53 45

3.3. Statistical analysis

*
Experimental design was conducted by means a two-

10,278
10,199

10,756
11,099

11,636
9720
3352

3364

3459
level half-fractional factorial design in order to evaluate

1
all single effects and second-order interactions between fac-
tors. Resultant design is shown in Table 2. A multiple com-

11,603
12,032

12,644
14,800

15,444
3695

3850

4322
10,987
PROT
parison analysis was carried out to assess significant
differences among the samples. Fisher’s least significant dif-

0
ferences (LSD) test was used to describe means with 95%
confidence. Data on instrumental texture parameters dur-

*
ing storage were evaluated by repeated measures analysis

10,483
10,483

11,056
11,429

11,984
9905
3440

3474

3549
of variance (ANOVA). The results obtained allowed estab-
lishing staling behaviour of enzyme-supplemented bread

1
crumb. Statgraphics Plus V5.1 and Statistica V6 programs

11,398
11,748

12,344
14,471

15,097
3607

3740

4231
10,803
were used as statistical analysis software.

XYL
0
4. Results and discussion

*
10,389
10,234

10,793
11,263

11,804
9805
3393

3403

3499
4.1. Dynamic viscoelastic properties of enzyme-supplemented
doughs

1
AMYL

11,491
11,996

12,608
14,637

15,277
3654

3811

4281
10,903
Individual effects of enzymes on dynamic moduli of
doughs are showed in Table 3. Except GO and LAC, all

0
enzymes affected significantly (p < 0.05) the rheological
behaviour of dough. TG and PROT modified dough rheol-

10,528
10,628

11,204
12,376

12,943
9958
3405

3474

3745
ogy at all studied resting periods. However AMYL and
1
XYL only had a significant effect on mentioned moduli
after 180 min of incubation. The addition of TG led to a
11,353
11,603

12,196
13,523

14,138
3642

3740

4036
10,750
LAC

significant increase in elastic (G 0 ), viscous (G00 ) and complex

0

(G*) moduli of doughs. These results were similar to those

obtained by previous investigations (Caballero et al., 2005;
10,618

11,210
11,239

11,823
13,344

13,958
3584

3626

4009
Single effects of design factors on viscoelastic properties of enzyme-supplemented doughs

Gujral & Rosell, 2004b; Kö ksel, Sivri, Ng, & Steffe, 2001;
1

Larre´ et al., 2000) and confirmed the strengthening action

The effect of the factor is significant with a significance level of 95% (p < 0.05).
exerted by TG due to its crosslinking effect on different
10,671
10,991

11,577
12,555

13,123
3463

3588

3771
10,090

flour protein fractions (Autio et al., 2005; Bauer et al.,

GO
0

2003a; Gerrard et al., 2001; Larre´ et al., 2000; Rosell

et al., 2003). All dynamic moduli showed an steady increase
*

*
*

with increasing incubation time, which proved the cumula-

tive effect of TG. PROT diminished significantly elastic (G 0 )
11,985

12,579
13,765

14,364
18,075

18,688
3806

4048

4763

and complex (G*) moduli, whereas decrease in viscous

modulus (G00 ) was only significant (p < 0.05) after a
1

180 min resting period. The weakening effect of PROT

8722
3241
9301
8466
3167
9036
7824
3018
8393
TGa

was also related with the decrease in resistance to extension

0

observed by Indrani, Prabhasankar, Rajiv, and Venkatesw-

See Table 2 for levels of design factors.

ara-Rao (2003). Proteinase activity affects specially to

Overall mean

glutenins (Bombara, Anon, & Pilosof, 1997), which would

10,354

10,940
11,115

11,700
12,950

13,540
3523

3607

3890

alter the elasticity of the gluten complex.

Both polysaccharide-degrading enzymes promoted a
similar significant decrease of all dynamic moduli analysed
when samples were incubated during 180 min. Mart´ınez-
Units

Anaya and Jimenez (1997, 1998) stated that hydrolytic

Pa
Pa
Pa
Pa
Pa
Pa
Pa
Pa
Pa

enzymes acting on carbohydrates induce a quick response

in dough rheology and their action continue during resting.
Parameter

Analysis of second-order interactive effects of design fac-

Table 3

G0180 min
G180 min
G30 0 min
G30 min
Gm30 min
G060 min
G60 min
Gm60 min

Gm180 min

tors (enzymes) on viscoelastic properties of dough revealed

significant (p < 0.05) interactions between TG and XYL,
00

00

00

*
a
 46
Table 4
Single effects of design factors on bread quality of enzyme-supplemented doughs

P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53

Parameter Units Overall TGa GO LAC AMYL XYL PROT
mean 0 1 0 1 0 1 0 1 0 1 0 1
* * * *
Height/width 0.87 0.87 0.86 0.84 0.90 0.86 0.87 0.84 0.89 0.84 0.89 0.81 0.92
ratio
Specific volume cm3/g 3.73 3.85 3.61 *
3.67 3.80 3.73 3.74 3.56 3.91 *
3.53 3.94 *
3.40 4.01 *
* * * *
Hardness gf 376 297 456 402 351 375 378 451 301 443 310 494 259
* *
Cohesiveness 0.83 0.82 0.84 0.82 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83
* * * *
Gumminess gf 312 242 382 330 293 310 313 374 250 367 256 408 216
* * * *
Chewiness gf 306 237 374 323 288 304 307 366 245 359 252 398 213
*
Springiness 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.99
* * *
Resilience 0.45 0.44 0.46 0.44 0.46 0.45 0.45 0.46 0.44 0.46 0.45 0.45 0.45
*
Crumb 160 151 169 160 160 159 161 159 161 159 162 163 158
brightness
*
Mean cell area mm2 1.48 1.78 1.18 1.49 1.46 1.50 1.45 1.41 1.54 *
1.44 1.52 1.33 1.63 *
* *
Cell density cells/ 30 23 37 30 31 31 30 31 30 30 31 34 27
cm2
* * * *
Void fraction % 41.5 42.8 40.2 41.0 40.2 41.4 41.6 40.5 42.5 40.7 42.3 40.1 42.9
*
Cell wall mm 0.75 0.81 0.69 0.76 0.73 0.76 0.74 0.76 0.74 0.77 0.73 0.73 0.77
thickness
* *
Grain 11.7 7.2 16.1 11.7 11.7 11.9 11.4 12.6 10.7 12.3 11.1 14.5 8.8
uniformity
a
See Table 2 for levels of design factors.
*
The effect of the factor is significant with a significance level of 95% (p < 0.05).
 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53 47

and between AMYL and PROT (data not shown). The
protein polymerisation promoted by TG counteracted the
softening effect of XYL after a large resting period. These
results were consistent with those obtained after individual
addition of both enzymes but disagreed with the synergistic
diminution of uni- and bi-axial extensibility by the combi-
nation of TG and XYL observed by Collar and Bollain
(2004).
4.2. Bread quality of enzyme-supplemented doughs
Bread quality parameters of doughs were significantly
(p < 0.05) affected by individual enzyme addition, except
when LAC was used (Table 4). The greater effect was
induced by TG, since this enzyme widely modified morpho-
metric, textural and crumb grain properties of fresh pan
breads. TG decreased significantly loaf specific volume
but did not produce changes in its shape. The strengthening
effect and dough extensibility reduction promoted by TG,
probably decreased dough extension during fermentation
and oven-spring. According to previous findings, the loaf
volume could be only increased when additional water
was applied (Autio et al., 2005), and when a poor baking
quality flour was used together with TG (Basman, Kö ksel,
& Perry, 2002). Single presence of TG led to a significant
increase of hardness, cohesiveness, gumminess, chewiness
and resilience of bread crumb. Crumb grain profile of TG-
supplemented breads showed brighter crumb, smaller cells,
greater cell density and grain uniformity, and smaller void
fraction and cell wall thickness. These results denote a finer
and more uniform overall structure, which is consis- tent
with an improved bread crumb grain (Sapirstein, 1999).
Similar textural and crumb grain profiles have been stated
previously by means of sensorial and instrumental studies
of breads prepared with TG (Basman et al., 2002; Bauer,
Koehler, Wieser, & Schieberle, 2003b; Collar & Bol-

Table 5
Second-order interactive effects of design factors on morphometric and textural properties of enzyme-supplemented fresh pan breads
Parameter Units Overall mean Levela TG/AMYL TG/XYL TG/PROT GO/PROT AMYL/PROT XYL/PROT
* * *
Height/width ratio 0.87 00 0.86 0.77 0.76
01 0.88 0.91 0.92
10 0.77 0.86 0.86
11 0.96 0.93 0.92
Specific volume cm3/g 3.73 00 3.73* 3.22*
01 3.97 4.11
10 3.06 3.57
11 4.17 4.02
Hardness gf 376 00 327* 318* 362* 625*
01 266 275 231 277
10 576 568 625 362
11 337 345 287 240
Cohesiveness 0.83 00
01
10
11

Gumminess gf 312 00 266* 260* 294* 516*

01 217 224 190 231
10 481 475 522 300
11 283 289 242 200
Chewiness gf 306 00 262* 254* 288* 503* 482*
01 212 220 187 228 236
10 470 464 509 293 314
11 278 284 239 198 190
Springiness 0.98 00
01
10
11
Resilience 0.45 00
01
10
11
a
See Table 2 for levels of design factors.
*
The effect of the factor is significant with a significance level of 95% (p < 0.05).
48 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53

lain, 2005a; Collar, Bollain, & Angioloni, 2005; Gerrard
et al., 1998).
GO-supplemented doughs yielded loaves with an
increased height/width ratio, characterized by more elastic
and cohesive crumbs. Polysaccharide-degrading enzymes
and PROT exercised similar suitable effects on pan bread
quality parameters. Their use led to better shape, greater
specific volume and void fraction of loaves. This behaviour
was more marked when PROT was added to dough, and
came accompanied by significant decreases in crumb hard-
ness, gumminess and chewiness. Additionally, PROT gave
more elastic crumb and a coarser bread crumb structure,
which was characterized by greater cells, less cell density
and more irregular cellular structure. Moreover, AMYL
also increased mean cell area and decreased crumb elastic-
ity. A more open gluten network formed by fibrous ele-
ments has been suggested by Blaszczak, Sadowska,
Rosell, and Fornal (2004) as the responsible for the higher
elasticity and lower hardness of the crumb after treatments
with AMYL.
Analysis of second-order interactive effects of design fac-
tors on bread quality parameters revealed significant
(p < 0.05) interactions between TG and all the other
enzymes except GO (Tables 5 and 6). LAC addition to
TG containing doughs only modified significantly crumb
grain features, yielding loaves with less crumb brightness
and cell density, but greater mean cell area and cell wall
thickness than those obtained by the treatment with singly
TG. Through simultaneous arabinoxylans gelation (Figue-
roa-Espinoza & Rouau, 1998) and oxidative action (Labat,
Morel, & Rouau, 2000), LAC promoted a finer crumb
structure than control samples. However, this enzyme
would favour the interference of pentosans in glutenins
aggregation (Primo-Mart´ın et al., 2003), modifying TG
strengthening effect and resulting in a coarser crumb.
Moreover, AMYL, XYL and PROT exerted a softener
effect on the crumb of TG-supplemented pan breads, lead-
ing to significant decreases in hardness, gumminess and
chewiness of samples. Interactive effect of TG and XYL
on bread quality could arise from rheological changes,
which were consistent, in turn, with the release of pento-
sans from gluten network (Primo-Mart´ın et al., 2003).
TG and PROT showed a significant synergistic effect on
height/width ratio and specific volume of loaves. Likewise,
PROT gave a more marked diminution of hardness and
related parameters than AMYL or XYL, exhibiting values
even lower than control samples. Crumb grain profile was
also significantly affected by TG/PROT interaction. PROT
addition increased void fraction and decreased grain uni-
formity of TG-treated samples. These results denoted that
the hydrolytic effect of PROT, probably counteracted
excessive protein polymerisation catalyzed by TG, making
possible a better dough development during fermentation
and oven-spring. Gottmann and Sproessler (1994) proved

Table 6
Second-order interactive effects of design factors on crumb grain characteristics of enzyme-supplemented fresh pan breads
Parameter Units Overall mean Levela TG/LAC TG/PROT LAC/XYL LAC/PROT AMYL/PROT
Crumb brightness 160 00 147* 155*
01 156 163
10 172 163
11 167 160
Mean cell area mm2 1.48 00 1.91* 1.42*
01 1.64 1.58
10 1.09 1.23
11 1.26 1.68
Cell density cells/cm2 30 00 20* 28*
01 26 34
10 41 32
11 34 28
Void fraction % 41.5 00 42.4* 37.9*
01 43.1 43.0
10 37.7 42.2
11 42.7 42.8
Cell wall thickness mm 0.75 00 0.87* 0.82*
01 0.75 0.70
10 0.65 0.72
11 0.73 0.75
Grain uniformity 11.7 00 8.3*
01 6.2
10 20.7
11 11.4
a
See Table 2 for levels of design factors.
*
The effect of the factor is significant with a significance level of 95% (p < 0.05).
 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53 49

an undesired loss of extensibility after TG addition, and
proposed its combination with a protease in order to avoid
it.
AMYL and PROT combination led to significant
improvement of loaf shape, although increase in height/
width ratio was the same to that individually promoted
by AMYL. Similar behaviour was observed in crumb void
fraction, which value was also substantially higher than the
one obtained for control samples. However, hardness,
gumminess and chewiness clearly showed another trend,
suggesting a significant synergistic effect of AMYL and
PROT combination. GO and PROT combined synergisti-
cally improved loaf height/width ratio and loaf specific vol-
ume. The enhancement of this parameter was comparable
with that obtained for singly PROT treatment.
LAC interacted significantly with PROT and XYL, to
produce changes that essentially affected to the crumb
grain pattern of loaves. LAC promoted a finer crumb
grain, whereas PROT addition gave greater cells. However,
the combined use of these enzymes led to a coarser struc-
ture, denoting a protein weakening effect. The interference
of pentosans in the aggregation of gluten due to LAC action
(Primo-Mart´ın et al., 2003), would prevail over
disulfide linkages promotion, inducing, in the presence of
PROT, gas cells coalescence phenomena. Simultaneous
supplementation with LAC and XYL gave rise to signifi-
cant effects on crumb brightness, cell density and cell wall
thickness.
4.3. Enzyme-supplemented bread staling during storage
Repeated measures analysis of variance enabled us to
establish the single and the second-order interactive effects
of the enzymes on the trend and extent of variation of
instrumental texture parameters of enzyme-supplemented
pan breads during the storage. Statistical analysis con-
firmed significant (p < 0.05) individual effects of the
enzymes TG, AMYL, XYL and PROT. TG significantly
affected to the evolution of all textural parameters in the
time. Bread staling increased by TG addition, and affected
specially to hardness (Fig. 1a), chewiness and gumminess.
These results differed from those obtained with enriched
formulation (Collar & Bollain, 2005a). Martin, Zeleznak,
and Hoseney (1991) suggested that interactions between
the swollen starch granules and the protein network
actively contribute to crumb firming. Through microscopic

2800 2800

2400 2400

2000 2000
HARDNESS (gf)

HARDNESS (gf)

1600 1600

1200 1200

800 800
WITH AMYL
400 WITH TG 400 WITHOUT AMYL
WITHOUT TG
0 0
0 2 5 8 12 0 2 5 8 12

a DAYS b DAYS

2800 2800

2400 2400

2000 2000
HARDNESS (gf)

HARDNESS (gf)

1600 1600

1200 1200

800 800

400 WITH XYL 400 WITH PROT

WITHOUT PROT
WITHOUT XYL
0 0
0 2 5 8 12 0 2 5 8 12

c DAYS d DAYS

Fig. 1. Significant single effects of design factors on crumb hardness evolution during storage of enzyme-supplemented pan breads [TG (a), AMYL (b),
XYL (c) and PROT (d)]. Vertical bars denote 0.95 confidence intervals for means. Continuous line represents the evolution of bread cru mb hardness in
presence of the factor, whilst discontinuous line represents the evolution of bread crumb hardness in absence of the factor (see Table 2 for codes of design
factors).
50 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53

4000 4000

3600 WITH TG 3600

3200 WITHOUT TG 3200

2800 2800
HARDNESS (gf)
2400 2400

2000 2000

1600 1600

1200 1200

800 800

400 400

0 0
5 8 12 0 2 5 8 12
0 2
Days Days

a WITH AMYL WITHOUT AMYL

0 2 5 8 12 0 2 5 8 12
4000 4000

3600 3600

3200 3200

2800 2800
HARDNESS (gf)

2400 2400

2000 2000

1600 1600

1200 1200

800 800

400 400

0 0 2 5 8 12 0
0 2 5 8 12
Days
Days
b WITH XYL
WITHOUT XYL

4000 4000

3600 3600

3200 3200

2800 2800
HARDNESS (gf)

2400 2400

2000 2000

1600 1600

1200 1200

800 800

400 400

0 0 2 5 8 12 0
0 2 5 8 12
Days
Days

c WITH PROT
WITHOUT PROT

4000 4000

3600 WITH AMYL 3600

WITHOUT AMYL
3200 3200

2800 2800
HARDNESS (gf)

2400 2400

2000 2000

1600 1600

1200 1200

800 800

400 400

0 0 2 5 8 12 0
0 2 5 8 12
Days
Days
WITH PROT
d WITHOUT PROT

Fig. 2. Significant second-order interactive effects of design factors on crumb hardness evolution during storage of enzyme-supplemented pan breads [TG/
AMYL (a), TG/XYL (b), TG/PROT (c) and AMYL/PROT (d)]. Vertical bars denote 0.95 confidence intervals for means (see Table 2 for codes of design
factors).
 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53
analysis of bread crumb, significant differences in starch–
protein matrix have been detected in the course of storage
(Blaszczak et al., 2004). TG-induced strengthening effect
could increase such interactions and favour bread staling
and simultaneous crumb elasticity preservation during
storage. The affinity to water promoted by TG in gluten
(Gerrard et al., 1998) could also limit the water availability
for starch and accelerate its retrogradation.
On the contrary, AMYL, XYL and PROT exhibited a
significant antistaling effect (Fig. 1b–d). PROT showed
the most marked effect on reducing hardness, which came
accompanied by a significant slowing down in gumminess
and chewiness evolution in the time (data not shown).
According with the conclusions of Armero and Collar
(1998), crumb firming during storage mainly depends on
initial crumb firmness. Therefore, softener effect of AMYL,
XYL and PROT (Fig. 1) would justify partially its influ-
ence on firming kinetics. Alpha-amylase has been proved
to be useful for reducing amylopectin retrogradation and
the firming rate of wheat bread crumb (Champenois, della
Valle, Planchot, Buleon, & Colonna, 1999) and rice bread
crumb (Gujral, Haros, & Rosell, 2003). Although Sahl- strö
m and Brathen (1997) indicated that the mechanisms
governing crumb firmness and the retrogradation of amy-
lopectin seemed to be different, Morgan, Gerrard, Every,
Ross, and Gilpin (1997) suggested that starch retrograda-
tion is sufficient to cause bread firming. Through studies
carried out on model systems, Rojas, Rosell, and Benedito
de Barber (2001) concluded that maltodextrins were
responsible for the antistaling effect promoted by addition
of a-amylase to bread formulation. They proposed the
exis-
tence of a mechanism of partial obstruction of starch retro-
gradation. Jiménez and Martı́nez-Anaya (2001) proved
that water-insoluble pentosans (WIP) were positively corre-
lated with crumb elasticity and hardness during storage.
XYL would lead to cleavage of the backbone of arabinoxy-
lans, with the consequent release of water and WIP diminu-
tion (Rouau, El Hayek, & Moreau, 1994), which could
explain the positive effects of XYL in bread freshness. Sim-
ilarly, the improvement of bread shelf-life through PROT
addition possibly would be tied with the increase of the
water available for starch, in conjunction with a simulta-
neous diminution of starch–protein interactions as conse-
quence of the hydrolysis of peptide bonds in the protein
molecules. Babiker, Fujisawa, Matsudomi, and Kato
(1996) previously reported an increase in the hydrophobic-
ity of protease-treated gluten.
Statistical analysis of the textural data during storage
proved significant (p < 0.05) second-order interactive
effects between enzymes. AMYL, XYL and PROT dimin-
ished significantly the staling effect promoted by TG. Their
action was showed clearly through crumb hardness evolu-
tion (Fig. 2a–c). However, the behaviour of these samples
did no reach to that of single AMYL, XYL and PROT-
supplemented breads. The mechanisms by which these
enzymes slowed down staling kinetics of TG-treated sam-
ples probably were rather different. Whilst XYL and
51
AMYL would act on dough polysaccharide fraction,
PROT directly would counteract TG-action, by simulta-
neously acting on dough protein fraction. Besides their
ability to modify the degree of protein polymerisation
and consequently, the starch–protein interactions, TG/
PROT combination has been reported as responsible for
changing the number of exposed hydrophobic residues
(Babiker et al., 1996), which could alter dough water avail-
ability. Using dynamic and static deformation measure-
ments, Bolla´ın, Angioloni, and Collar (2005) confirmed
synergistic interactions regarding staling behaviour of
breads formulated with TG/XYL and TG/AMYL combi-
nations. Addition of bacterial alpha-amylase to TG-sup-
plemented proved to significantly slow down the staling
kinetics determined as cohesiveness and resilience (Collar
& Bollain, 2005a).
AMYL and PROT also combined synergistically to
decrease bread staling during storage, as could be deduced
from their significant effect on crumb firming kinetics
(Fig. 2d).
5. Conclusions
Among all gluten crosslinking enzymes analysed,
dynamic rheological test only showed a significant single
effect of transglutaminase. Protease decreased dynamic
moduli at all studied resting periods, whilst polysaccha-
ride-degrading enzymes modified dough rheology after
180 min of incubation. Statistical analysis of viscoelastic
properties revealed that simultaneous use of TG and
XYL could be an interesting alternative for avoiding exces-
sive dough strengthening promoted by TG.
Bread quality parameters of doughs were significantly
affected by individual enzyme addition, except when LAC
was used. TG provided the greatest effect, since this enzyme
widely modified morphometric, textural and crumb grain
properties of fresh pan breads. Polysaccharide-degrading
enzymes and PROT led to better shape, greater specific vol-
ume and void fraction of loaves. Except GO, all enzymes
showed significant interactive effects with TG. In accor-
dance with crumb hardness evolution, it was proved that
AMYL, XYL and PROT were able to diminish the staling
effect promoted by TG. AMYL and PROT also combined
synergistically to decrease bread firming during storage.
Therefore, the antistaling effect of PROT was confirmed.
Likewise, results suggest that, through different mecha-
nisms, dough protein and polysaccharide fractions actively
contribute to bread staling kinetics.
Acknowledgements
This work was financially supported by Comisión Inter-
ministerial de Ciencia y Tecnolog´ıa Projects (MCYT,
AGL2002-04093-C03ALI and AGL2005-05192-C04),
Consejo Superior de Investigaciones Cient´ıficas (CSIC)
and Universidad de Valladolid, Spain. The authors would
like to thank R. Mart´ınez (Novo Nordisk, Madrid, Spain)
52 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53
for providing enzyme samples. Likewise, authors thank
Beatriz Barcenilla, Margarita Fernandez and Sonia Mart´ın
for their collaboration in this study. Part of this work has
been awarded with the 4th Research Prize CETECE (Fun-
dación Centro Tecnológico de Cereales de Castilla y León).
References
AACC (2000). Approved methods of the AACC. St. Paul, Minnesota:
American Association of Cereal Chemists.
Armero, E., & Collar, C. (1998). Crumb firming kinetics of wheat breads
with anti-staling additives. Journal of Cereal Science, 28(2), 165–174.
Autio, K., Kruus, K., Knaapila, A., Gerber, N., Flander, L., & Buchert, J.
(2005). Kinetics of transglutaminase-induced cross-linking of wheat
proteins in dough. Journal of Agricultural and Food Chemistry, 53(4),
1039–1045.
Babiker, E. E., Fujisawa, N., Matsudomi, N., & Kato, A. (1996).
Improvement in the functional properties of gluten by protease digestion
or acid hydrolysis followed by microbial transglutaminase treatment.
Journal of Agriculture and Food Chemistry, 44, 3746–3750. Basman, A.,
Kö ksel, H., & Perry, K. W. N. (2002). Effects of increasing levels of
transglutaminase on the rheological properties and bread quality of two
wheat flours. European Food Research and Technology,
215, 419–424.
Bauer, N., Koehler, P., Wieser, H., & Schieberle, P. (2003a). Studies of the
effects of microbial transglutaminase on gluten proteins of wheat I:
biochemical analysis. Cereal Chemistry, 80(6), 781–786.
Bauer, N., Koehler, P., Wieser, H., & Schieberle, P. (2003b). Studies of the
effects of microbial transglutaminase on gluten proteins of wheat II:
rheological properties. Cereal Chemistry, 80(6), 787–790.
Blaszczak, W., Sadowska, J., Rosell, C. M., & Fornal, J. (2004).
Structural changes in the wheat dough and bread with the addition of
alpha-amylases. European Food Research and Technology, 219(4),
348–354.
Bolla´ın, C., Angioloni, A., & Collar, C. (2005). Bread staling assessment
of enzyme-supplemented pan breads by dynamic and static deforma-
tion measurements. European Food Research and Technology, 220(1),
83–89.
Bolla´ın, C., & Collar, C. (2004). Dough viscoelastic response of
hydrocolloid/enzyme/surfactant blends assessed by uni- and bi-axial
extension measurements. Food Hydrocolloids, 18(3), 499–507.
Bombara, N., Anon, M. C., & Pilosof, A. M. R. (1997). Functional
properties of protease modified wheat flours. Lebensmittel Wissens-
chaft und Technologie, 30(5), 441–447.
Bonet, A., Caballero, P. A., Gomez, M., & Rosell, C. M. (2005).
Microbial transglutaminase as a tool to restore the functionality of
gluten from insect-damaged wheat. Cereal Chemistry, 82(4), 425–
430.
Bonet, A., Rosell, C. M., Caballero, P. A., Gomez, M., Pérez-Munuera, I.,
& Hernando, I. (2006). Glucose oxidase effect on dough rheology and
bread quality: a study from macroscopic to molecular level. Food
Chemistry. doi:10.1016/j.foodchem.2005.07.43.
Caballero, P. A., Bonet, A., Rosell, C. M., & Gomez, M. (2005). Effect of
microbial transglutaminase on the rheological and thermal properties
of insect damaged wheat flour. Journal of Cereal Science, 42(1), 93–
100.
Caballero, P. A., Gomez, M., & Rosell, C. M. (2006). Bread quality and
dough rheology of enzyme-supplemented wheat flour. European Food
Research and Technology, in press, doi:10.1007/s00217-006-0311-3.
Champenois, Y., della Valle, G., Planchot, V., Buleon, A., & Colonna, P.
(1999). Influence of alpha-amylases on bread staling and on retrogra-
dation of wheat starch models. Sciences des Aliments, 19(3–4), 471–
486.
Collar, C., & Bollain, C. (2004). Impact of microbial transglutaminase on
the viscoelastic profile of formulated bread doughs. European Food
Research and Technology, 218(2), 139–146.
Collar, C., & Bollain, C. (2005a). Impact of microbial transglutaminase on
the staling behaviour of enzyme supplemented pan breads. European
Food Research and Technology, 221(3–4), 298–304.
Collar, C., & Bollain, C. (2005b). Relationships between dough functional
indicators during breadmaking steps in formulated samples. European
Food Research and Technology, 220(3–4), 372–379.
Collar, C., Bollain, C., & Angioloni, A. (2005). Significance of microbial
transglutaminase on the sensory, mechanical and crumb grain pattern
of enzyme supplemented fresh pan breads. Journal of Food Engineer-
ing, 70(4), 479–488.
Figueroa-Espinoza, M. C., Morel, M. H., & Rouau, X. (1998). Effect of
lysine, tyrosine, cysteine, and glutathione on the oxidative cross-
linking of feruloylated arabinoxylans by a fungal laccase. Journal of
Agriculture and Food Chemistry, 46(7), 2583–2589.
Figueroa-Espinoza, M. C., & Rouau, X. (1998). Oxidative cross-linking of
pentosans by a fungal laccase and horseradish peroxidase: mechanism
of linkage between feruloylated arabinoxylans. Cereal Chemistry,
75(2), 259–265.
Gerrard, J. A., Fayle, S. E., Brown, P. A., Sutton, K. H., Simmons, L., &
Rasiah, I. (2001). Effects of microbial transglutaminase on the wheat
proteins of bread and croissant dough. Journal of Food Science, 66(6),
782–786.
Gerrard, J. A., Fayle, S. E., Wilson, A. J., Newberry, M. P., Ross, M., &
Kavale, S. (1998). Dough properties and crumb strength of white pan
bread as affected by microbial transglutaminase. Journal of Food
Science, 63(3), 472–475.
Goesaert, H., Brijs, K., Veraverbeke, W. S., Courtin, C. M., Gebruers, K.,
& Delcour, J. A. (2005). Wheat flour constituents: how they impact
bread quality, and how to impact their functionality. Trends in Food
Science and Technology, 16(1-3), 12–30.
Gottmann, K., & Sproessler, B. (1994). Baking agent and process for the
manufacture of doughs and bakery products. European Patent
Application EP0492406, B1.
Gujral, H. S., Haros, M., & Rosell, C. M. (2003). Starch hydrolyzing
enzymes for retarding the staling of rice bread. Cereal Chemistry,
80(6), 750–754.
Gujral, H. S., & Rosell, C. M. (2004a). Improvement of the breadmaking
quality of rice flour by glucose oxidase. Food Research International,
37, 75–81.
Gujral, H. S., & Rosell, C. M. (2004b). Functionality of rice flour modified
with a microbial transglutaminase. Journal of Cereal Science, 39, 225–
230.
Hoseney, R. C., & Faubion, J. M. (1981). A mechanism for the oxidative
gelation of wheat flour water soluble pentosans. Cereal Chemistry, 58,
421–424.
Indrani, D., Prabhasankar, P., Rajiv, J., & Venkateswara-Rao, G. (2003).
Scanning electron microscopy, rheological characteristics, and bread-
baking performance of wheat-flour dough as affected by enzymes.
Journal of Food Science, 68(9), 2804–2809.
Jiménez, T., & Martı́nez-Anaya, M. A. (2001). Amylases and hemicellu-
lases in breadmaking. Degradation by-products and potential rela-
tionship with functionality. Food Science and Technology International,
7(1), 5–14.
Köksel, H., Sivri, D., Ng, P. K. W., & Steffe, J. F. (2001). Effects of
transglutaminase enzyme on fundamental rheological properties of
sound and bug-damaged wheat flour doughs. Cereal Chemistry, 78(1),
26–30.
Kuraishi, C., Yamazaki, K., & Susa, Y. (2001). Transglutaminase: its
utilization in the food industry. Food Reviews International, 17(2),
221–246.
Labat, E., Morel, M. H., & Rouau, X. (2000). Effects of laccase and ferulic
acid on wheat flour doughs. Cereal Chemistry, 77(6), 823–828.
Labat, E., Morel, M. H., & Rouau, X. (2001). Effect of laccase and
manganese peroxidase on wheat gluten and pentosans during mixing.
Food Hydrocolloids, 15(1), 47–52.
Larré, C., Denery, P. S., Popineau, Y., Deshayes, G., Desserme, C., &
Lefevre, J. (2000). Biochemical analysis and rheological properties of
gluten modified by transglutaminase. Cereal Chemistry, 77(1), 32–38.
 P.A. Caballero et al. / Journal of Food Engineering 81 (2007) 42–53
Martin, M. L., Zeleznak, K. J., & Hoseney, R. C. (1991). A mechanism of
bread firming. I. Role of starch swelling. Cereal Chemistry, 68(5),
498–503.
Mart´ınez-Anaya, M. A., & Jimenez, T. (1997). Rheological properties of
enzyme supplemented doughs. Journal of Texture Studies, 28(5), 569–
583.
Mart´ınez-Anaya, M. A., & Jimenez, T. (1998). Physical properties of
enzyme-supplemented doughs and relationship with bread quality
parameters. Zeitschrift fu¨ r Lebensmittel Untersuchung und Forschung,
206(2), 134–142.
Morgan, K. R., Gerrard, J., Every, D., Ross, M., & Gilpin, M. (1997).
Staling in starch breads: the effect of antistaling alpha-amylase. Starch,
49(2), 54–59.
Motoki, M., & Nio, N. (1983). Crosslinking between different food
proteins by transglutaminase. Journal of Food Science, 48(2), 561–566.
Motoki, M., & Seguro, K. (1998). Transglutaminase and its use for food
processing. Trends in Food Science and Technology, 9, 204–210.
Poulsen, C., & Hostrup, P. B. (1998). Purification and characterization of
a hexose oxidase with excellent strengthening effects in bread. Cereal
Chemistry, 75(1), 51–57.
Primo-Mart´ın, C., Valera, R., & Mart´ınez-Anaya, M. A. (2003). Effect of
pentosanase and oxidases on the characteristics of doughs and the
glutenin macropolymer (GMP). Journal of Agricultural and Food
Chemistry, 51, 4673–4679.
53
Rojas, J. A., Rosell, C. M., & Benedito de Barber, C. (2001). Role of
maltodextrins in the staling of starch gels. European Food Research and
Technology, 212(3), 364–368.
Rosell, C. M., Wang, J., Aja, S., Bean, S., & Lookhart, G. (2003). Wheat
flour proteins as affected by transglutaminase and glucose oxidase.
Cereal Chemistry, 80(1), 52–55.
Rouau, X., El Hayek, M. L., & Moreau, D. (1994). Effect of an enzyme
preparation containing pentosanases on the bread-making quality of
flours in relation to changes in pentosan properties. Journal of Cereal
Science, 19(3), 259–272.
Sahlström, S., & Brathen, E. (1997). Effects of enzyme preparations for
baking, mixing time and resting time on bread quality and bread
staling. Food Chemistry, 58(1–2), 75–80.
Sapirstein, H. D. (1999). The imaging and measurement of bubbles in
bread. In G. M. Campbell, C. Webb, S. S. Pandiella, & K. Niranjan
(Eds.), Bubbles in food (pp. 233–243). St. Paul, Minnesota: American
Association of Cereal Chemists.
Vemulapalli, V., & Hoseney, R. C. (1998). Glucose oxidase effects
on gluten and water solubles. Cereal Chemistry, 75(6), 859–
862.
Zhu, Y., Rinzema, A., Tramper, J., & Bol, J. (1995). Microbial
transglutaminase: a review of its production and application in food
processing. Applied Microbiology and Biotechnology, 44(3–4), 277–
282.